Since the DNA shuffling technique developed by Stemmer was published in Science in 1994, many molecular breeding technologies relating to the artificial evolution of genes have been developed (Juha P. Int Arch Allergy Immunology. 121, 173-182 (2000)), including various improved protocols of gene shuffling (Huimin Z. et al. Nucleic Acids Research, 25 (6), 1307-1308 (1997); Andreas C. et al. Nature, 391 (15), 288-291 (1998); Miho K. et al. Gene, 236, 159-167 (1997)), staggered extension process (Huimin 7. et al. Nature Biotechnology, 16, 258-261 (1998)), incremental truncation for the creation of hybrid enzymes (Marc 0. et al. Nature Biotechnology, 17, 1205-1209 (1999)) and random chimeragenesis on transient templates (Wayne M C. et al. Nature Biotechnology, 19, 354-359 (2001)) etc. To date there are many successful examples in which the basic principles of the molecular evolution techniques have been applied to generate or modify genes in fields ranging from the common biological proteases to improvement of antibiotic titre, the degradation of pollutants in the environment, the reconstruction of viruses, and the development of pharmaceuticals. But it is rarely employed in field of DNA vaccines which is the third generation of human vaccines. Although many experts predict that the success of gene shuffling technology in gene vaccines will make it widely applicable to diseases such as cancer, autoimmune diseases and infectious diseases which severely harm human health (Dewey D. Y. R et al. Biotechnology Progress, 16 (1), 2-16 (2000); Phillip A P. et al. Current Opinion in Biotechnology, 8, 724-733 (1997); Robert G. W. et al. Curr Opin Mol Ther, 3 (1), 31-36 (2001)), there is no related literature or patents demonstrating substantial progress.
Gene vaccines represent a new immunological theory and technique developed in the 1990s and are the third generation of vaccines after attenuated virus vaccines and subunit vaccines (Wolff J. A. et al. Science, 247, 1465-8 (1990)). The technology of gene vaccines comprises the step of direct injection of plasmid DNA containing exogenous protein coding sequences into the body so as to enable the direct expression of the exogenous proteins in the body thereby eliciting an immune response. Gene vaccines have many advantages compared to conventional vaccines, such as prolonged immune response, simultaneous induction of humoral immunity and cytotoxic T cell response, simple preparation, convenience, inexpensive, stable antigen and convenient delivery, and so on. It not only has the safety proved by recombinant subunit vaccines and the high efficiency of attenuated virus vaccines for the induction of a general immune response but also elicits specific types of immune response in the body. Up to now, gene vaccines have been widely used for therapy of infectious diseases and cancer caused by viruses, bacteria and protozoa as well as in the therapy of allergic response and tolerance in new born infants. There is beneficial development in the therapy against influenza, AIDS, rabies, hepatitis B, tuberculosis, malaria and leishmaniosis (Lai W. C. et al. Crit. Rev Immunol, 18 (5), 449-84 (1998)). With respect to plasmodium, HIV and other highly variable viruses severely affecting human health, there are no very effective vaccines. For pathogens with highly variable properties, vaccine studies indicate that it is necessary to employ various antigens at various periods (Doolan D. L. et al. Int J Parasitol, 31 (8), 753-62 (2001)). For multiple antigen vaccines, many reports and patents have been published, which focus on single synthetic or recombinant vaccines of multiple antigens and polyepitope protein vaccines or the combination of limited types of such synthetic or recombinant vaccines. Moreover, it is problem that the synthesis of polypeptide vaccines is very costly, which hampers its practical application. Now some references report polyepitope chimeric gene vaccines, but the attention concentrates on the artificial and single chimeric pattern among polyepitope genes, and no immunoprotective effect better than that of polypeptide vaccine has been achieved. In view that three antigenic epitopes of Plasmodium falciparum (MSA-1, NKND and CST3) were selected during construction of a multivalent recombinant DNA vaccine, the inventors of the present invention carried out different construction and combination of polyepitope genes according to the combination pattern designed in advance and found that there was an optimal assembly in polyepitope combination (Lin C. T. Chinese J of Biochemistry and Molecular Biology, 1999, 15 (6): 974-977). The result indicated that with respect to the combination of a few epitopes (less than 3), the optimal combination may be obtained by manually individual assembly and construction. But as the combination of more epitopes (more than 3) provides many possibilities, it is impractical to assemble and construct by the above method because it is complicated, costly and requires much work. Thus, how to effectively design polyepitope genes and overcome the variability of pathogens is required for the development of gene vaccines (Yu Z. et al. Vaccine, 16 (7), 1660-7 (1998); Kumar S. et al. Trends Parasitol, 18 (3), 129-35 (2002); Hoffman S. L. et al. Dev Biol, 104, 121-32 (2000); Li M. et al. Chin Med J (Engl), 112 (8), 691-7 (1999); Jiang Y. et al. Chin Med J (Engl), 112 (8), 686-90 (1999)).
The life cycle of Plasmodium falciparum which causes malignant malaria severely affecting human health is complicated and comprises four stages comprising asexual reproduction and sexual reproduction in humans and sexual reproduction and sporogony in mosquitoes. In humans there are exoerythrocytic (liver) and erythrocytic stages, while gametocyte and sporozoite stages are in mosquitoes. Such complex biological traits cause Plasmodium falciparum to have highly variable response against the immunoprotection of the host and drugs, so that single protective antigenic vaccines against malaria are not effective.
The clinical symptoms caused by plasmodium are mainly due to its asexual reproduction in the red blood cells of the host. Erythrocytic stage vaccines are designed to act directly against this unique pathogenic stage of plasmodium. Malaria vaccines comprise attenuated circumsporozoite vaccine, subunit vaccine and synthetic peptide vaccine, but they are not successful because the various antigens against which various vaccines are directed can not generate satisfactory protective effects. Therefore, it is well accepted in the art that the combination of multi-stage and multivalent epitopes is necessary in the construction of a malaria vaccine, to make it possible to obtain the desired protective effect. However, it is difficult to determine the quantity and linking order of the genes encoding polypeptides during the construction of multi-stage and multivalent vaccines manually, and the induction of humoral immunity by epitope DNA vaccines is generally not satisfactory, which are problems to be solved.